Polynucleotides for multivalent RNA interference, compositions and methods of use thereof

Abstract

The present invention includes bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and/or in which the target regions are complementary to more than one target gene or target sequence. Also included are compositions comprising such nucleic acid molecules and methods of using the same for multivalent RNA interference and the treatment of a variety of diseases and infections.

The invention claimed is: 
     
       1. A method of reducing expression of one or more target genes, comprising introducing a polynucleotide complex into a cell, the cell comprising the one or more target genes, wherein the polynucleotide complex consists of:
 a first polynucleotide that is fully or partially complementary to a first target sequence, wherein the first target sequence is a region of the one or more target genes, and wherein the first polynucleotide is approximately 15-30 nucleotides in length; 
 a second polynucleotide that is fully or partially complementary to a second target sequence, wherein the second target sequence is a region of the one or more target genes, and wherein the second polynucleotide is approximately 15-30 nucleotides in length; and 
 a third polynucleotide that is either (a) fully or partially complementary to a third target sequence, wherein the third target sequence is a region of the one or more target genes, or (b) not specific to any target sequence, and wherein the third polynucleotide is approximately 15-30 nucleotides in length; 
 wherein a 3′ region of the first polynucleotide is complementary to a 5′ region of the second polynucleotide, where a 3′ region of the second polynucleotide is complementary to a 5′ region of the third polynucleotide, and wherein a 3′ region of the third polynucleotide is complementary to a 5′ region of the first polynucleotide; and 
 wherein the first, second, and third polynucleotides hybridize via the complementary 3′ and 5′ regions to form a polynucleotide complex with a first, second, and third stem region 
 and wherein the introduction of the polynucleotide complex to the cell results in a reduction in expression of the one or more target genes. 
 
     
     
       2. The method of  claim 1 , wherein the polynucleotide complex is introduced to the cell using a method selected from transfection, lipofection, scrape-loading, electroporation, microinjection, infection, gene gun, and retrotransposition. 
     
     
       3. The method of  claim 1 , wherein the cell is a plant cell, an animal cell, a protozoan cell, a bacterial cell, or a fungal cell. 
     
     
       4. The method of  claim 3 , wherein the animal cell is a mammalian cell selected from a human cell, a murine cell, a rodent cell, and a primate cell. 
     
     
       5. The method of  claim 1 , wherein the cell is a cancer cell, a virally infected cell, a mutated cell, or a normal cell. 
     
     
       6. The method of  claim 1 , wherein the cell is derived from primary cells, a cell line, immortalized cells, or transformed cells. 
     
     
       7. The method of  claim 1 , wherein the one or more target genes comprise a gene, an mRNA, a microRNA, or a combination thereof. 
     
     
       8. The method of  claim 1 , wherein the one or more target genes comprise an oncogene, a mutated gene, an mRNA transcript of a viral gene, or an mRNA transcript of a human apolipoprotein B (ApoB) gene. 
     
     
       9. A method of reducing gene expression of one or more target genes comprising introducing a self-hybridizing polynucleotide into a cell, the cell comprising the one or more target genes, wherein the self-hybridizing polynucleotide consists of a single polynucleotide molecule comprising:
 a first target-specific region that is fully or partially complementary to a first target sequence, wherein the first target sequence is a region of the one or more target genes, and wherein the first target-specific region is approximately 15-30 nucleotides in length; 
 a second target-specific region that is fully or partially complementary to a second target sequence, wherein the second target sequence is a region of the one or more target genes, and wherein the second target-specific region is approximately 15-30 nucleotides in length; and 
 a third target-specific region that is either (a) fully or partially complementary to a third target sequence wherein the third target sequence is a region of the one or more target genes, or (b) not specific to any target sequence, and wherein the third target-specific region is approximately 15-30 nucleotides in length; 
 wherein a 3′ region of the first target-specific region is complementary to a 5′ region of the second target-specific region within the single polynucleotide molecule, where a 3′ region of the second target-specific region is complementary to a 5′ region of the third target-specific region within the single polynucleotide molecule, and wherein a 3′ region of the third target-specific region is complementary to a 5′ region of the first target-specific region within the single polynucleotide molecule; and 
 wherein at least one of the first, second or third target-specific region binds or hybridizes to another target-specific region of the same single polynucleotide molecule via the complementary 3′ and 5′ regions to form at least one stem region and at least one loop or stem loop 
 and wherein the introduction of the polynucleotide complex to the cell results in a reduction in the expression of the one or more target genes. 
 
     
     
       10. The method of  claim 9 , wherein the self-hybridizing polynucleotide is encoded by a vector. 
     
     
       11. The method of  claim 9 , wherein the vector is introduced to the cell using a method selected from transfection, lipofection, scrape-loading, electroporation, microinjection, infection, gene gun, and retrotransposition. 
     
     
       12. The method of  claim 9 , wherein the cell is a plant cell, an animal cell, a protozoan cell, a bacterial cell, or a fungal cell. 
     
     
       13. The method of  claim 12 , wherein the animal cell is a mammalian cell selected from a human cell, a murine cell, a rodent cell, and a primate cell. 
     
     
       14. The method of  claim 9 , wherein the cell is a cancer cell, a virally infected cell, a mutated cell, or a normal cell. 
     
     
       15. The method of  claim 9 , wherein the cell is derived from primary cells, a cell line, immortalized cells, or transformed cells. 
     
     
       16. The method of  claim 9 , wherein the one or more target genes comprise a gene, an mRNA, a microRNA, or a combination thereof. 
     
     
       17. The method of  claim 9 , wherein the one or more target genes comprise an oncogene, a mutated gene, an mRNA transcript of a viral gene, or an mRNA transcript of a human apolipoprotein B (ApoB) gene.